Pediatric healthcare providers and parents find it challenging to identify rare health conditions that can present themselves in the neonatal period due to lack of suitable tests. Many of these conditions are rare genetic disorders which are typically suspected due to abnormal newborn screening results, clinical symptoms or as a concern within families with a history of genetic abnormalities. For example fatty acid oxidation disorder (an IEM) may have generic symptoms like lethargy, vomiting, and liver anomalies, which make it very hard to pinpoint the condition in the newborn. Annually, 13,000 neonates in the USA are suspected to be affected by different inborn errors of metabolism (IEM) and 3-6% newborns have rare genetic syndromes. It is extremely challenging to precisely determine by a single test the specific disorder from amongst thousands of disorders, especially where simple analytes are unavailable. Effective treatment of genetic conditions can prevent outcomes such as mental retardation, brain injury, or sudden death. The current approach of analyte specific screening does not provide the genetic etiology and the comprehensive coverage of disorders. The single gene sequencing method used as a last resort for confirming is costly ($200-2000) and time consuming (12-16 weeks turnaround). Such DNA sequencing test is also impractical because it requires significant blood volume that exposes an infant to risk from blood loss. We are developing a test focused on these disorders using massively parallel DNA sequencing (Next Generation Sequencing (NGS) methodology. It can address the deficiencies of current testing and be used as a routine primary or second-tier newborn screening tool, first in high-risk patients and later in pre-symptomatic patients. Accurate pre-symptomatic diagnosis is a prerequisite for effective disease prevention and management. Newborn Disease Panel-based NGS screening and diagnostics is currently unavailable from a commercial company. Such a commercial NGS-based test with broad coverage of such disorders can be rapidly expanded throughout the country as a service, permitting quick diagnostic decisions. Using this framework, we can improve child health, eliminate protracted and costly medical interventions, and discern key relationships between gene, environment and disease. Our first Specific Aim is to make this test accurate by examining variability over an entire genic region and development of workflows on whole or dried blood specimens. In our second Specific Aim, we will focus on a comprehensive gene panel tested on 50 different genetic variations in a single test, and compare our method with conventional DNA and another NGS sequencing method. The resulting prototype will be a valuable first step towards a NGS based screening and diagnostic test that will address the needs of these high-risk infants. It will help healthcare providers, patients, and families to understand the precise cause of metabolic disorders and genetic syndromes, and to remove uncertainty and delay. Our test will simultaneously screen and diagnose hundreds of genetic conditions at once from a single sample, providing comprehensive information at an affordable price.